CD34 is a surface glycophosphoprotein that is expressed only on developmentally early hematopoietic progenitor cells (HPC), small vessel endothelial cells, embryonic fibroblasts and possibly brain and testis parenchymal cells. Its expression is lost as HPC cells differentiate into mature blood cells, indicating a potential regulatory role in early hematopoiesis. Indeed a subset of patients with Acute Myelogenous Leukemia which contain highly undifferentiated CD34+ blast cells are more resistant to induction chemotherapy, suggesting that CD34 positivity may be a negative risk factor for treatment of certain cancer cells. However, in spite of the experimental and potential clinical utility of anti-CD34 antibodies (eg. Myl0) that can identify and purify early HPC's for study, the role and function of CD34 is unknown. Recently we have discovered two apparently different mechanisms involved in CD34 regulation. Our results suggest a possible causative role for PKC activation and direct phosphorylation of CD34 in the rapid up-regulation of CD34 surface expression. We have also identified a second TPA mediated mechanism, occurring hours after TPA addition, which accounts for the downregulation of steady state CD34 mRNA levels followed by decreased surface expression in hematopoietic cells. This proposal aims to determine the role and function of CD34 in developmental hematopoiesis and to elucidate the mechanism(s) of regulation of CD34 expression. Furthermore, our results should answer the question of whether CD34 is expressed on the hematopoietic stem cell. We will employ state of the art molecular and biochemical methodology, including creation of heterozygous (+/-) and homozygous (-/-) CD34 gene "knock-outs" in embryonic stem (ES) cells that will be useful to study the effect on developmental hematopoiesis both in vitro and, by producing transgenic mice with these mutated ES cells, in vivo. Our results are expected to help fill in fundamental gaps in knowledge regarding the physiologic function and regulation of this important early developmental surface protein.